Phospholipid formulations and uses thereof in lung disease detection and treatment

ABSTRACT

Disclosed are methods and compositions that are useful in the detection and therapy of diseases (e.g., emphysema) and damage that afflict the lungs. In some aspects, the compositions comprise a formulation enriched for a species of phosphatidylcholine, such as palmitoylmyristoyl phosphatidylcholine (16:0/14:0PC). The compositions may further be described as lung surfactant supplement preparations particularly useful in the treatment of pulmonary diseases and afflictions prevalent among premature infants, and in particular, Respiratory Distress Syndrome (RDS). A PC marker is also disclosed, 16:0/14:0PC, that may be used to detect pulmonary disease or reduced/compromised alveolar function in an animal. Phospholipid profiles of 16:0/14:0PC, 16:0/16:1PC and 16:0/16:0PC are also provided, and are correlated with particular pulmonary diseased states.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application makes reference to Provisional U.S. Patent ApplicationSer. No. 06/676,949, filed May 3, 2005. The entire disclosure andcontents of the above application is hereby incorporated by reference.

BACKGROUND

1. Field of the Invention

The present invention relates generally to the field of surfactantpreparations and surfactant supplements for the lung. The invention alsorelates to the field of lung disease detection and treatment, as amarker of lung disease comprising a characteristic phospholipid profileis presented, and is correlated with specific developmental anddisease-related changes in lung tissue.

2. Related Art

Pulmonary surfactant is a complex mixture of lipids and proteins that issynthesized and secreted by alveolar type II epithelial cells. Thesecells secrete this mixture of lipids and proteins into the thin liquidlayer that lines the epithelium. Once in the extracellular space,surfactant reduces surface tension at the air-liquid interface of thelung, a function that requires an appropriate mix of surfactant lipidsand the hydrophobic proteins, surfactant protein (SP)-B and SP-C (1, 2).Of the surfactant lipids, 80-90% are phospholipids, with the rest beingneutral lipids. The most abundant phospholipid species isphosphatidylcholine (PC) with dipalmitoyl-PC (16:0/16:0PC) beingimportant in attaining acceptable surface tensions (near 0 mN/m (3-7)).Although saturated palmitoylmyristoyl-PC (16:0/14:0PC) andmonounsaturated palmitoylpalmitoleoyl-PC (16:0/16:1PC) are alsoprevalent in most mammalian lung surfactants (7), their contribution tosurfactant function is not well understood. In vitro studies havedemonstrated that dipalmitoyl-PC (16:0/16:0PC) does not spread well atthe air/liquid interface (8, 9). One possibility is thatpalmitoylmyristoyl-PC (16:0/14:0PC) and palmitoylpalmitoleoyl-PC(16:0/16:1PC) assist in the surface spreading of dipalmitoyl-PC(16:0/16:0PC) (10, 11).

The phospholipids palmitoylmyristoyl-PC (16:0/14:0PC) andpalmitoylpalmitoleoyl-PC (16:0/16:1PC) may contribute to dynamicsurfactant functions during mammalian respiration (4). Fractionalconcentrations of both of these PC species in lung surfactant have beenfound to correlate with respiratory rates in mammals (4). Withincreasing respiratory rates, both palmitoylmyristoyl-PC (16:0/14:0PC)and palmitoylpalmitoleoyl-PC (16:0/16:1PC) concentrations in surfactantare increased. Thus, the fractional concentrations ofpalmitoylmyristoyl-PC (16:0/14:0PC) and palmitoylpalmitoleoyl-PC(16:0/16:1PC) in surfactant are adapted to the physiological needs ofthe mammalian lung (4).

There are two events during lung development that may have specificsurfactant needs. First, at birth, the lungs require large amounts ofsurfactant to convert the fluid-filled airspaces into gas-exchange unitswith a stable air/liquid interface. Failure to establish a low surfacetension air/liquid interface at the distal airspace results inrespiratory distress syndrome (RDS), a relatively common complication ofpremature birth as a result of delayed lung fluid clearance and/orpulmonary surfactant insufficiency (12, 13). Second, in severalmammalian species distal lung development proceeds postnatally, suchthat the alveoli are formed exclusively after birth (e.g. rats and mice)or predominantly after birth (e.g. humans).

The process of alveolarization involves the division of the preexistingvoluminous terminal air sacs (saccules) into smaller units, the alveoli,by secondary septa. These septa grow out from the saccular walls intothe air spaces in a centripetal manner. As a result there is an increasein the number of terminal gas exchange units (14). Although these newlyformed alveoli have less volume there is a substantial net increase intotal surface area (14). In rats (14) and mice (15), the bulk ofsecondary septation takes place between postnatal days 4 and 14. Humanalveolarization occurs mainly between 36 weeks of gestation and 18months of age (16). The completion of alveolarization results in anincreased number of terminal airway units which continue to grow in sizeto further expand surface area into adult life. Whether thesemorphological surface changes during alveolarization impact on thecomposition of functionally important surfactant PC species is alsounknown.

The state of the art demonstrates that a medical need continues to existfor preparations that are effective and useful for enhancing assessmentof lung development and alveolarization, especially in monitoring andtreating premature infants. In addition, a need continues to exist for areliable marker for assessing lung disease and lung developmentalstages, particularly as related to alveolarization, such that anappropriate therapeutic preparation, such as a surfactant preparationand/or other therapeutic intervention may be made available to thepatient.

SUMMARY

The present invention in an overall and general sense relates to thecharacterization and discovery of particularly defined phospholipidpreparations having significance in the identity of pathological anddevelopmental abnormalities in the lung, and particularly to the healthand development of alveolar tissue in the lung.

The present invention provides a variety of pharmaceutically acceptablepreparations of specific formulations of phospholipid PC preparations.These preparations may be formulated for delivery to a patient in needthereof according to a variety of techniques well known to those ofskill in the formulary and pharmaceutical arts.

The invention also provides a reliable marker for alveolar pathology,and as such provides a clinical indicator of reduced alveolarization orpulmonary disease state in a subject animal. This marker comprises aparticular phospholipid, palmitoylmyristoyl-PC (16:0/14:0PC). Therelative concentration of this phospholipid species in a subject animalsample, such as a lung sample, has been correlated by the presentinventors with an increased incidence of a variety of pulmonary diseasesand conditions of compromised pulmonary function in infants and adults.In particular embodiments, a reduced relative concentration ofpalmitoylmyristoyl-PC (16:0/14:0PC) in a subject animal sample, inparticular a lung sample, relative to a control animal sampleconcentration of palmitoylmyristoyl-PC (16:0/14:0PC), is diagnostic ofreduced alveolarization of the lung, possibly related to pulmonarydisease or exposure to a toxic substance. In particular embodiments ofthe method, the concentration of palmitoylmyristoyl-PC (16:0/14:0PC) isreduced at least 20%, 25%, 40%, or as much as 90% to 100%, relative to acontrol sample palmitoylmyristoyl-PC (16:0/14:0PC) concentration, inanimals having pulmonary disease or having been exposed to a toxic solidor air-born chemical or pollutant.

By way of example, representative lung diseases and pathologies forwhich the presently disclosed marker and preparations may be used toidentify and treat include, but are not limited to, emphysema,respiratory distress syndrome (both adult and infant), idiopathicpulmonary fibrosis, bronchopulmonary dysplasia (chronic lung disease),asthma, and congenital malformations (eg. lung hypoplasia, congenitallobar emphysema, congenital cystic adenomatoid malformations, congenitalalveolar capillary dysplasia, alphal antitrypsin deficiency and others),lung ischemia-reperfusion injury (LIRI), chronic lung disease (CLD) andmeconium aspiration syndrome (MAS). The marker may also be used topredict risk for development of more advanced forms of pulmonarydistress/damage. For example, a correlation is known to exist betweenrelative 16:0/14:0 concentration of an infant sample lung sample and toa heightened incidence for the development of respiratory distress. Thisparticular prognostic indicator for predicting risk of progressive lungdisease finds clinical application for use in a method for assessingrisk for developing more serious or continued pulmonary disease in aninfant having respiratory distress. A heightened risk of developingprogressively more severe pulmonary distress exists particularly in aninfant maintained or having been maintained on a pulmonary respirator orother assisted breathing apparatus.

A diagnostic PC profile has also been identified and correlated with adiseased or compromised pulmonary state in an animal resulting frompulmonary disease or exposure to a toxic substance or environmentallycompromising event (high O₂, extreme pressure changes, etc.). Inparticular embodiments, the PC profile comprises the animal's relativelung sample palmitoylmyristoyl-PC(16:0/14:0PC) concentration. An animallung sample having a sample palmitoylmyristoyl-PC (16:0/14:0PC) lessthan a level of palmitoylmyristoyl-PC(16:0/14:0PC) in a control animallung sample is diagnostic of a diseased pulmonary state, a reduced orcompromised alveolarization state in the animal, or of exposure to apulmonary toxic substance. Such a diagnostic use may find application insettings where humans have been or are exposed to potentiallycompromising inhaled substances. Examples of such potentiallycompromising inhaled substances include by way of example, and notlimitation, air-born industrial and environmental chemicals (smog),smoke, inhaled steroids (such as particular inhaled asthma medicaments,“puffers”), dexamethasone, chemical waste products, alcohols, and thelike.

In some embodiments, the PC profile characteristic of pulmonary exposureto a toxic or potentially toxic substance, such as dexamethasone,comprises a measure of the total phospholipid PC content of a subjectanimal lung sample. A PC profile of this nature in an animal having beenexposed to a toxic substance would be elevated relative to the total PCcontent of a control animal lung sample. The total PC content may befurther defined as comprising a measure of the lung samplepalmitoylpalmitoleoyl-PC (16:0/16:1PC) and palmitoyloleoyl-PC(16:0/18:1PC) content. The total PC measure in an animal having beenexposed to a toxic substance will be elevated about 10% to about 20%, ormore, over total PC content observed from lung tissue of a controlanimal. In particular embodiments, the relative total PCcontent/concentration may be used as part of a method for detectingpulmonary damage or reduced pulmonary function resulting from toxin orchemical exposure, such as from exposure to dexamethasone.

In other embodiments, the total PC content of an animal lung sample maybe used as part of a method to detect or diagnose exposure to high O₂concentrations. In these embodiments, the total PC content comprises ameasure of palmitoylpalmitoleoyl-PC (16:0/16:1PC) and palmitoyloleoyl-PC(16:0/18:1PC). The total PC content will be reduced in a subject animalthat had been exposed to O₂, relative to a control animal lung sampletotal PC content/concentration. The amount/concentration of total PC inan O₂ exposed animal lung sample will be reduced 20%, 40%, or even more,compared to a control animal lung sample.

In other embodiments, the PC profile in a diseased or pulmonarycompromised animal may be detected through a measure of thedipalmitoyl-PC (16:0/16:0PC) concentration of the animal lung sample. Inthis context, an elevated (about 20% to about 40%) concentration ofdipalmitoyl-PC (16:0/16:0PC) compared to a control animal lung sample,is diagnostic of diseased or compromised pulmonary function.

A diagnostic PC alveolar content profile is specifically defined foranimals having reduced alveolar function as a result of disease, such asin emphysema, respiratory distress syndrome (infant and adult), andother disease states related to lung and pulmonary function. Thisprofile includes a reduced relative concentration of lung tissuepalmitoylmyristoyl-PC (16:0/14:0PC). A method employing this profile todiagnose and detect disease is provided. In one aspect, the methodcomprises obtaining a lung sample from a subject animal to provide asubject animal lung sample, measuring the amount ofpalmitoylmyristoyl-PC (16:0/14:0PC) in the subject animal lung sample,and comparing the amount of palmitoylmyristoyl-PC (16:0/14:0PC) in thesubject animal lung sample to an amount ofpalmitoylmyristoyl-PC(16:0/14:0PC) in a control animal lung sample,wherein a reduced concentration of palmitoylmyristoyl-PC (16:0/14:0PC)in the subject animal lung sample relative to the concentration ofpalmitoylmyristoyl-PC (16:0/14:0PC) in the control animal lung sample isdiagnostic of reduced alveolar function/architecture or alveolar damageattendant disease.

The animal samples that may be analyzed and used for the variousdiagnostic, therapeutic and forensic applications described herein mayconstitute an infant, fetal, adult, or even cadaver harvested lungtissue specimen. The preparations, markers, and methods of the inventionare suitable for both human and veterinary use, and therefore findsapplication for use in humans, domestic animals (horses, cats, dogs,pigs), and other commercially valuable animal species (monkeys, lambs, ,rats, mice, hamsters, guinea pigs, bears, deer, cows, chickens, etc.).

The present invention also provides a number of surfactant andsurfactant supplement preparations tailored to treat and manage lungdisease and compromised alveolar/pulmonary function in a newborn/infant(to 18 months). These particular surfactant and surfactant supplementsof the invention employ the correlation established by the presentinventors between the absolute and fractional changes in concentrationsof functionally important PC species (dipalmitoyl-PC (16:0/16:0PC),palmitoylmyristoyl-PC (16:0/14:0PC) andpalmitoylpalmitoleoyl-PC(16:0/16:1PC) ) immediately after birth. Forexample, several unique developmental stage dependent PC profiles areidentified here, and used to formulate custom tailored phospholipidsurfactant preparations and surfactant supplements that will enhancepostnatal lung development and deter/inhibit damage to alveolar tissue.

The specific compositional amount of each of these 16:0/16:1PC,16:0/14:0PC and 16:0/16:0PC species that will be in each formulationwill vary with the specific developmental stage and pathology of thesubject being treated. In some embodiments, the specific composition ofthe PC formulations will be enriched for the PC species identified to bedeficient in a subject animal. One such surfactant PC formulationtailored for use in infants with surfactant deficiency is enriched forpalmitoylmyristoyl-PC (16:0/14:0PC). By way of example, such aformulation would include a concentration of about 20% to about 50%fractional concentration of palmitoylmyristoyl-PC (16:0/14:0PC). Anothersuch surfactant PC formulation may be enriched forpalmitoylpalmitoleoyl-PC (16:0/16:1PC) for use in newborns immediatelyor shortly after birth to enhance surfactant spreading capacities. Byway of example, such a formulation would include a concentration ofabout 20% to 40% fractional concentration of palmitoylpalmitoleoyl-PC(16:0/16:1PC). By way of reference, conventional surfactantpreparations, such as Curosurf®, contain 80 mg/ml phospholipids, ofwhich 70% are phosphatidylcholine. About 30% of the phosphatidylcholineshould be palmitoylpalmitoleoyl-PC (16:0/16:1PC) which is about 15-20mg/ml. An average treatment dosage of Curosurf® administered to anewborn infant is about 100-200 mg/kg per dose.

The surfactants and surfactant supplements may also be used as a carrierfor therapeutically, prophylactically, and/or diagnostically suitable oractive substance or substances, e.g., pulmonary drug delivery.

The invention also provides a pharmaceutical kit. In some embodiments,the kit comprises a container means comprising a preparation enrichedfor 16:0/14:0PC, 16:0/16:1PC, 16:0/18:0PC, 16:0/18:1PC, or a particulardesired mixture thereof, and a second container means comprising asuitable pharmaceutical grade carrier solution. This carrier solutionwill be suitable for suspending or dissolving the contents of the firstcontainer means to provide a preparation of the desired concentration ina ready to use form for the subject patient. It is envisioned that foruse in an infant having RDS, the preparation will be enriched for16:0/14:0PC. The kit may optionally also include an instructional sheet.This instructional sheet may include instructions on how the PC is to bereconstituted depending upon the particular use for which it will bemade, directions for administration, recommended dosages, storingconditions, and appropriate warnings. The kit may also include a devicefor facilitating administration of the preparation to the subject, suchas a tracheal tube, aspirator, or other appropriate device.

The surfactants and surfactant supplements of the invention may alsoinclude surfactant proteins, such as SP-A, SP-B, SP-C, SP-D, or mixturesthereof. The palmitoylmyristoyl-PC (16:0/14:0PC), 16:0/16:1PC,16:0/16:0PC, 16:0/18:0PC and 16:0/18:1PC of the surfactants andsurfactant supplements may be of synthetic origin and obtained fromother than a porcine or bovine tissue source. Some embodiments of thesurfactants and surfactant supplements may be prepared from phospholipidspecies obtained or derived from porcine or bovine tissue origin, orobtained from recombinant cells engineered to express the appropriatedesired ingredient.

In particular embodiments, the surfactant and surfactant supplements areformulated so as to be suitable for delivery through a tracheal tubeinto the lungs of a patient subject animal. In other embodiments, theformulation may be prepared so as to be suitable for delivery as anaerosol. These and other delivery forms are readily prepared for use inthe practice of the present invention given the specific types andratios of specific phospholipids described herein, and those formulationtechniques known to those in the formulary arts, such as are describedin Remington's Pharmaceutical Sciences (61), which text is specificallyincorporated herein by reference.

The following abbreviations are used through out the description of theinvention:

-   16:0/14:0PC—Palmitoylmyristoyl Phosphatidylcholine;-   16:0/16:0PC—Dipalmitoyl Phosphatidylcholine;-   16:0/16:1PC—Palmitoylpalmitoleoyl Phosphatidylcholine;-   16:0/18:1PC—Palmitoyloleoyl Phosphatidylcholine;-   ALI—Acute Lung Injury;-   ARDS—Adult Respiratory Distress Syndrome;-   BALF—Bronchoalveolar Lavage Fluid;-   BPD—Broncho pulmonary Dysplasia;-   CLD—Chronic Lung Disease;-   COPD—Chronic Obstructive Pulmonary Disease;-   DPPC—Dipalmitoyl Phosphatidylcholine (16:0/16:0PC) (also known as    colfosceril palmitate);-   EMO—Extracorporeal Membrane Oxygenation;-   IPF—Idiopathic Pulmonary Fibrosis;-   IRDS—Infant Respiratory Distress Syndrome;-   LIRI—Lung Ischemia-Reperfusion Injury;-   MAS—Meconium Aspiration Syndrome;-   PC—Phosphatidylcholine;-   RDS—Respiratory Distress Syndrome;-   SP—Surfactant Protein.

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in conjunction with the accompanyingdrawings, in which:

FIGS. 1(a)-1(c): Developmental profile of total PC: (1 a) Total PCcontent in bronchoalveolar lavage fluid (BALF) of untreated rats. (1 b)Total PC content of BALF during late fetal gestation. (1 c) Total PCcontent of BALF during first 4 days after birth. Mean±SE, n=4 animalsper time point [except for samples taken in the first 20 minutespostpartum (n=3 animals) and day 10 (n=8 animals)]. * P<0.01.

FIGS. 2(a)-2(d): Concentration of three major PC species inbronchoalveolar lavage fluid during rat development: (2 a)dipalmitoyl-PC (16:0/16:0PC), (2 b) palmitoylpalmitoleolyl-PC(16:0/16:1PC), (2 c) palmitoylmyristoyl-PC (16:0/14:0PC). (2 d) Thethree individual PC species presented as percent of total PC. Mean±SE,n=4 animals per time point [except for samples taken in the first 20minutes postpartum (n=3 animals) and day 10 (n=8 animals)].

FIGS. 3(a)-3(c): Percentage changes of the three major PC species inbronchoalveolar lavage fluid in mice and rats at late fetal gestation(mouse E18 (embryonic day 18), rat E22), at peak of alveolarization(postnatal day (PN) day 10, mice; PN14, rat), and mature juveniles(PN22). Mice (white bars) and rats (black bars). (3 a) dipalmitoyl-PC(16:0/16:0PC), (3 b) palmitoylpalmitoleolyl-PC (16:0/16:1PC), (3 c)palmitoylmyristoyl-PC (16:0/14:0PC). Individual species are presented aspercent of total PC. Mean±SE, n=4 animals per time point [except formice sample E18 (n=10) and PN 22 (n=10)]. * P<0.01.

FIGS. 4(a)-4(h): Bronchoalveolar lavage content of total PC (4 a, 4 e),dipalmitoyl-PC (16:0/16:0PC) (4 b, 4 f), palmitoylpalmitoleolyl-PC(16:0/16:1PC) (4 c, 4 g), palmitoylmyristoyl-PC (16:0/14:0PC) (4 d, 4h). Panels a-d from 10 day old rats. White bars: control rats; greybars: rats exposed to 60% O₂ and black bars: dexamethasone treated rats.Panels 4e-4h from 4 to 14 day old rats. Solid line: control rats anddashed line: rats exposed to 60% O₂. Mean±SE, n=4 animals per treatmentand time point. * P<0.01.

FIGS. 5(a)-5(b): Bronchoalveolar lavage and tracheal aspirate content of(5 a) dipalmitoyl-PC (16:0/16:0PC), (5 b) palmitoylmyristoyl-PC(16:0/14:0PC) from infants and adults with and without alveolarpathology. Mean±SE, RDS, n=19; CLD, n=8; lung tumor, n=8; transplant,n=4; COPD, n=3; IPF, n=3. * P<0.01.

FIGS. 6(a)-6(c): Correlations between palmitoylmyristoyl-PC(16:0/14:0PC) and alveolar curvature. (6 a) Developmental changes ofmean alveolar radius and palmitoylmyristoyl-PC (16:0/14:0PC)concentration in bronchoalveolar lavage. Alveolar radii were calculatedfrom published data of Burri et al.(14), divided in half (open circles),and from Blanco et al. (55), for day 2, 23, 40, and Blanco et al., (54)for day 14, 60, considering the form of alveoli as a sphere(Radius=3_(rd) root of [3× mean alveolar volume/4π]) (full circles,solid line). BALF concentration of palmitoylmyristoyl-PC (16:0/14:0PC)are shown as percent of total PC. (6 b) Light scattering ofspontaneously formed liposomes from BALF lipids. A correlation plot ofthe percentage of palmitoylmyristoyl-PC (16:0/14:0PC) in the BALF versusthe radius of spontaneously formed liposomes is shown. Mean±SE, n=4separate samples. (6 c) Developmental changes of dipalmitoyl-PC(16:0/16:0PC) (solid line) and palmitoylmyristoyl-PC (16:0/14:0PC)(dashed line) content in alveolar type II epithelial cells.

FIG. 7: Percent of 16:0/18:1 obtained from a normal rat profile.Developmental profile of palmitoyloleoyl-PC (16:0/18:1PC) concentrationin BALF samples from rat lung.

FIG. 8 Comparison of commercially available surfactant compositions,Survanta®, Curosurf®, and BLES. Phospatidylcholine concentrations in 3natural surfactants (Survanta®, Curosurf® and BLES) preparations andtracheal aspirate samples from 3-day newborn human (3 days postpartum)(white bar) and 35—week gestation (solid black bar) human infants.

FIG. 9: Prognostics indications of tracheal aspirate 16:0/14:0PCconcentrations in human infants for development of bronchopulmonarydysplasia (or chronic lung disease). A—Samples taken from patientsdiagnosed with RDS that did not develop BPD (CLD); B—Samples taken frompatients diagnosed with BPD; and C—Samples taken from patients diagnosedwith RDS that did not develop BPD (CLD).

DETAILED DESCRIPTION

It is advantageous to define several terms before describing theinvention. It should be appreciated that the following definitions areused throughout this application.

For administration by inhalation, compounds of the present invention canbe delivered in the form of aerosol spray presentation from pressurizedpacks or a nebulizer, with the use of a suitable propellant. In the caseof a pressurized aerosol, the dosage unit can be determined by providinga valve to deliver a metered amount. The formulation would be preparedas a powder for administration by inhalation. A powder form is obtained,for example, by mixing liquid lung surfactant preparations, for exampleaqueous suspensions, with aqueous suspensions of an enrichedconcentration of the palmitoylpalmitoleoyl-PC (16:0/16:1PC) and/orpalmitoylmyristoyl-PC (16:0/14:0PC) and/or dipalmitoyl-PC (16:0/16:0PC),alone or in combination with other desired ingredients, and then subjectto drying procedures whereby the liquid component is removed.Administration by inhalation can also be carried out by atomizingsolutions or suspensions which contain the compositions according to theinvention.

The compositions according to the invention may also be formulated in aliquid form for intratracheal or intrabronchial administration, or foruse as a lung wash/lavage fluid.

All of the formulations and additives of the invention may be preparedby procedures familiar to those skilled in the art, if appropriate usingfurther suitable pharmaceutical auxiliaries. Compositions according tothe invention advantageously contain the species of PC preparationsdescribed herein, and in particular an enriched concentration of thepalmitoylpalmitoleoyl-PC (16:0/16:1PC), palmitoylmyristoyl-PC(16:0/14:0PC) and/or dipalmitoyl-PC (16:0/16:0PC), alone or incombination with other desired ingredients, e.g. surfactant proteins.

Definitions

Where the definition of terms departs from the commonly used meaning ofthe term, applicant intends to utilize the definitions provided below,unless specifically indicated.

It should be noted that the singular forms, “a”, “an” and “the” includereference to the plural unless the context as herein presented clearlyindicates otherwise.

A “lung sample” is defined, by way of example, as a lung lavage sample,a lung tissue aspirate, a lung tissue biopsy, a fetal tissue biopsy, avillus tissue sample, a tracheal aspirate, a sputum sample, inducedsputum sample, or any other pulmonary derived tissue sample thatincludes an adequate amount of surfactant or of alveolar or other lungtissue cells or combination of cells sufficient to approximate therelative phospholipid content and/or mixture of phospholipids speciescontent. Hence, the sample should provide sufficient tissue needed toextract the component phospholipids in the sample.

The technique that will typically be used to analyze lipid content in ananimal lung sample is by a mass spectral analysis of lipids extractedfrom the subject lung sample. Yet another technique that may be used toquantify and qualitate phospholipid species in a tissue sample has beendescribed in Bernhard et al., Am. J. Respir. Crit. Care Med. 2004, 170(1):54-8, which is specifically incorporated herein by reference. Thistechnique may be used to specifically quantify surfactant PC synthesisin vivo. This method employs deuteriated choline coupled withelectrospray ionization tandem mass spectrometry. Advantages associatedwith both mass spectral techniques include the ability to accuratelyidentify and quantitate several different phospholipids at once in atest sample.

The compositions and other biological factors may be administeredthrough any known means. Alveolar administration, such as in aninhailable or aerosol formulation, provides an efficient approach forproviding and delivering the preparation to the tissue site in needthereof, such as the lungs.

An “enriched concentration” of a particular, for example, phospholipidspecies, as described herein is defined as a concentration of thespecified phospholipid that is greater than any other phospholipidspecies in the preparation. For example, where a formulation isdescribed as an “enriched 16:0:14:0PC” formulation, the preparation willinclude a greater concentration of the phospholipid 16:0/14:0PC, thanany other species of phosopholipid in the preparation. By way ofexample, this particular formulation may include a phospholipidcomponent that comprises at least about 50% 16:0/14:0PC (w/w) of thetotal phospholipid content of the formulation. An “enriched”phospholipid may comprise between 50% to about 99% of the specifiedphospholipid, relative to the total phospholipid component of theformulation. This percentage may also vary as between about 60% to about90%, about 75% to about 98%, about 80% to about 95%, about 85% to about95%, or even about 98% to 99% (w/w) of the phospholipid species. The“enriched” formulations may also be essentially free of any otherphospholipid species other than the species of phospholipid for which ithas been enriched. One such embodiment of the present formulations isessentially free of any other phospholipid other than 16:0:14:0PC.

A “therapeutically effective amount” of an active agent or combinationof agents as described herein is understood to comprise an amounteffective to elicit the desired response, but insufficient to cause atoxic reaction. A desired response, for example, may constitute theformation of a sufficient and/or acceptable alveolar film layer ofphospholipids at the desired air/tissue interface. The dosage andduration of treatment of the preparation to be administered to a subjectwill be determined by the health professional attending the subject inneed of treatment, and will consider the age, sex, weight, extent ofexisting alveolar development and/or damage of the subject, and specificformulation of phospholipids being used as treatment for the subject.

EXAMPLES

The following non-limiting examples are illustrative of the presentinvention, and should not be construed to constitute any limitation ofthe invention as it is described in the claims appended hereto.

Example 1 Materials and Methods

The present example describes the experimental protocols used tocharacterize the 16:0/14:0PC-enriched and other phospholipidpreparations described herein. This example also sets forth theprotocols that were employed in examining the developmental stages inthe lung, and the activity of the present preparations on the specificdevelopmental stages in the lung, especially those during earlypostnatal life. This example also sets forth the procedures that wereused to examine phospholipid profiles in human adult lungs, such as thatcharacteristic of adult human lungs in a patient with emphysema.

Animal Models—

Timed-pregnant female Wistar rats and C57/B16 mice were obtained fromCharles River (St. Constant, Qc, Canada). All animal protocols were inaccordance with Canadian Counsel of Animal Care guidelines and wereapproved by the Animal Care and Use Committee of the Hospital for SickChildren. Developmental profile: At fetal day 19 and 22 (term=23 days)the pregnant rat was anesthetized and the pups delivered by cesareansection, while postpartum rodents were removed from their motherdirectly before sacrifice. Dexamethasone treatment: Newborn Wistar ratswere injected daily for 4 days with dexamethasone (Sabex, Boucherville,Quebec, Canada) diluted in isotonic saline starting at postnatal day 1according to previously described protocols (17). Hyperoxia exposure:The oxygen treatment was performed by housing the rat pups with theirmother in chambers containing either 21% or 60% oxygen, starting at day1 until day of lavage (i.e. postnatal day 4, 7, 10 or 14) (18).

Bronchoalveolar Lavage Fluid Collection—

After sacrificing the animals, a needle (blunted tip) was insertedthrough a tracheostomy and the lungs were lavaged with a buffer composedof phosphate-buffered saline augmented with 0.05 mg/ml of 70 kDadextran-FITC [Molecular probes, Burlington, ON, Canada]. The inertfluorescent marker was included to determine lavage recovery (see FIG. 4e).

Tracheal Aspirate and Bronchoalveolar Lavage Fluid Collection—

Tracheal aspirates from infants were obtained by irrigation through theendotracheal tube using 1 ml of saline. Respiratory distress syndrome(RDS) was defined as a requirement for exogenous surfactant at the timeof birth for babies 27 weeks gestational age or older. For babies below27 weeks gestational age, RDS was defined as the ongoing need formechanical ventilation following exogenous surfactant therapy. Althoughthe diagnosis was made at the time of birth, the samples were collectedfrom 29 to 42 weeks corrected gestational age (median: 31 weeks; n=19).Bronchopulmonary dysplasia (BPD) was defined by a consistent clinicalcourse and x-ray changes. This was later confirmed by a continuedrequirement for supplemental oxygen at 36 weeks corrected gestationalage. The samples of BPD patients were collected between 28 and 43 weeksof gestation (median: 32 weeks; n=8). Prenatal steroids (Celestone,Schering, Berlin Germany) were administered to 21 of 28 patients,equally distributed between the RDS and BPD groups. For adult patientsbronchoscopy was done using 50 ml of saline for BAL. Samples fromemphysematic patients (n=3) and patients with idiopathic pulmonaryfibrosis (n=3) were collected prior to lung transplantation.

Control samples were taken prior to transplantation from patients withno alveolar complications (n=8) and from patients with lung tumors (n=4)with little or no chronic obstructive pulmonary disease (COPD). Sampleswere spun at 1000 g for 5 minutes to remove cellular material. Allpatient samples were obtained in accordance with Health Canada'sResearch Ethics Board guidelines.

Mass Spectral Analysis of PC—

BALF samples were spiked with 1 μg of deuterated dipalmitoyl-PC(16:0/16:0PC) (Avanti polar lipids, Alabaster Ala., USA) as an internalstandard, and then extracted (19). Lipids were analyzed using an API4000mass spectrometer [MDS SCIEX, Concord, Ontario, Canada) (20).

Light Scattering—

BALF samples, containing 50 nM of PC, were extracted and lipids weredried under nitrogen. Lipid samples were then reconstituted in 1 ml ofsaline at 37° C. followed by bath sonication. Following one freeze-thawcycle the vesicle size was determined by dynamic light scattering (21)using a Malvern Mastersizer X (Malvern Instruments Ltd., Worcestershire,United Kingdom).

Laser Capture Microdissection—

Cryo-embedded lung sections were processed as previously described (20).Alveolar Type II epithelial cells were visualized using a rabbitpolyclonal antibody against pro-NSP-C (private source) followed by aFITC-conjugated secondary goat anti-rabbit IgG antibody (Calbiochem, SanDiego, Calif., USA). Approximately 200 alveolar Type II epithelial cellswere captured using a PixCell II System (Arcturus, Mountain View,Calif., USA), lipids extracted and analyzed by mass spectrometry.

Statistics—

All values are shown as mean±standard error (SE). Statistical analysiswas done by Student's t-test or, for comparison of more than two groups,by one-way analysis of variance followed by Duncan's multiple rangecomparison test, with significance defined as P<0.05.

Example 2 Preparation of Phospholipid Surfactant Formulations,Pharmaceutical Preparations and Kits

The present example describes the methods by which the varioussurfactant preparations and lung surfactant replacement preparations maybe formulated for use according to the present invention. However, it isto be understood that other practical and well known formulationtechniques known to those of skill in the art may also be used, giventhe teaching provided herein of the specific types of phospholipids, thephosphatidylcholines and ratios of the phosphatidylcholines that aredemonstrated to have particular and specific activity for enhancingpulmonary function. In addition, those of skill in the art willrecognize appropriate variations from the procedures and reactionconditions specifically described herein, as well as substitutions forthe specific chemical reagents and components that may be used, inaccord with the practice of the present invention.

Formulation 1: Palmitoylpalmitoleoyl Phosphatidylcholine (16:0/16:1PC)

The use of this formulation is seen in situations e.g. where theestablishment of a first or new pulmonary surfactant film is needed. Byway of example, representative situations might be the establishment ofthe first surfactant film directly after birth in premature babies withsurfactant deficiency, in situations of altered surfactantfunction/quantity e.g. after meconium aspiration or pneumonia or afterinhalation/aspiration of toxic substances.

A preparation intended for this use will include an enrichedconcentration of 16:0/16:1PC. In particular applications, the amount of16:0/16:1PC will comprise at least 50% or more, and even up to 95% to100%, of the total PC content in the formulation.

Formulation 2: Palmitoylmyristoyl Phosphatidylcholine (16:0/14:0PC)

The use of this formulation is seen in situations e.g. where regularformation of the alveoli is inhibited or the architecture of healthyalveoli is disturbed. Hence, the particular architectural features ofthe pathological lung needs a particular fractional composition of thedifferent PC species discussed herein, in particular an enhancedconcentration of palmitoylmyristoyl-PC (16:0/14:0PC). By way of example,representative uses and situations include use in premature babies withprimary or secondary decreased alveolarization, BPD as an example.Furthermore all situations with secondary destruction of alveoli leadingto enlarged distal gas exchange units, e.g. emphysema, may be treatedusing these formulations enriched for palmitoylmyristoyl-PC(16:0/14:0PC).

Compositions and Pharmaceutical Kits:

In another aspect, the invention provides a pharmaceutical kitcomprising a first and a second container means, the first containermeans comprising a lung surfactant composition of PC, and in particularpalmitoylmyristoyl-PC (16:0/14:0PC), according to the invention and thesecond container means comprising a dispersion medium for the lungsurfactant composition. The lung surfactant composition may be in powderor particulate form. Any of the above individual or combination of PCformulations may be included in the kit first container means comprisingthe lung surfactant composition.

The PC-containing pharmaceutical compositions of the invention may be inpowder or particulate form adapted to be dispersed in an aqueous mediumbefore use. For example, the pharmaceutical compositions of PC of thekit may be in solid (e.g. powder, particles, granules, sachets, tablets,capsules etc.), semi-solid (gels, pastes etc.) or liquid (solutions,dispersions, suspensions, emulsions, mixtures etc) form and adapted foradministration via e.g. the respiratory organs. A pharmaceuticalcomposition in liquid form may be in the form of a dispersion comprisingthe lung surfactant composition and an electrolyte solution such as,e.g. a composition that is adapted to physiological conditions e.g. aphysiologically acceptable solution.

The pharmaceutical composition surfactant or surfactant supplement ofthe kits or individually provided products may further comprise anothertherapeutically, prophylactically and/or diagnostically activesubstance.

The pharmaceutical kit according to the present invention may includeinstructions with recommendations for the time period during which thelung surfactant composition should be administered after dispersion inthe dispersion medium.

Example 3 Characterization of Changing Phospholipid Profile DuringGestation and Early Postpartum Development

The present example is provided to demonstrate the utility of thepresent invention using total PC content in a subject lung sample as atool in identifying the pulmonary developmental stage and anyabnormalities thereof in an animal during gestation and early life (lessthan 18 months postpartum). The characteristic phospholipid profile maybe used to identify and diagnose pulmonary developmental abnormalities,and hence aid in the identification of a suitable treatment regimen forthe subject animal.

Bronchoalveolar lavage was performed on rats at differing gestationaland postpartum ages. Total PC concentration was determined by the sum ofthe concentrations of all individual PC species. As can be seen in FIG.1 a, PC content in BALF varied tremendously during fetal and postnatallung development. The amount of extracellular surfactant PC increasedsignificantly (40-fold) between 19 and 22 days' gestation (FIG. 1 b). Afurther 10-fold increase in total PC content of BALF occurred within thefirst two hours after birth (FIG. 1 c).

This immediate rise in surfactant PC following birth may be attributedto two factors. Firstly, the decrease in alveolar fluid via clearancewould concentrate the components of the bronchoalveolar compartment,including surfactant. At or shortly before birth the lung switches froma fluid secreting to a fluid absorbing organ. Although much of the lungfluid is cleared within 2 hours of birth there is evidence that theprocess of lung fluid adsorption is a more protracted process lastingmore than 40 hours in the rat (23). Secondly, it is known thatmechanical forces increase surfactant secretion (24-26). Lung expansiondue to the first deep sigh or onset of breathing has been shown tostimulate the release of preformed lamellar bodies into theextracellular space (27, 28). The concentration of PC in BALF decreasedslightly after 2 hours postpartum, but then increased and reachedmaximal levels at 24 hours after birth after which it declined andreached mature levels at day 4 postpartum (FIG. 1 c). While notintending to be limited to any particular mechanism of action or theory,this second postnatal surge in PC content in BALF may at least in partbe attendant the secretion of newly synthesized surfactant PC.

Earlier observations show that choline incorporation into rat lungtissue PC peaked on the first day after birth (see review (22)). Thesynthesis of new lamellar bodies has been estimated to requireapproximately 6 hours (29, 30). This may at least in part explain or berelated to the phenomenon observed of a plateau between the two peaks at2 and 24 hours postpartum. Thus, the requirement for surfactant duringearly extrauterine life is met by a release of preformed lamellar bodieswithin the first few hours of breathing, followed by massive synthesisand secretion by alveolar type II epithelial cells of new surfactantmaterial.

Alternatively, the second peak could be a secretion from a more slowlyreleased surfactant pool (31). How alveolar type II epithelial cellssense this need for new surfactant within 2 hours after the onset ofbreathing is unknown.

Another increase of total PC content in BALF occurred after postnatalday 4 with a peak at day 12 and a subsequent decline until day 19 whenadult levels were reached (FIG. 1 a). In rats, the bulk ofalveolarization takes place between days 4 and 14 (14). The concomitantrelationship between PC concentration and alveolarization identified inthe present invention has never been reported.

The enlargement in surface area that occurs during alveolarization wouldrequire an increasing amount of surfactant. The increase in the amountof surfactant PC during alveolarization suggests that there may be aco-regulation of surfactant and septal formation. Alveolar type IIepithelial cell numbers are believed to peak during alveolarization(32). The increased number of type II cells could account for increasedsurfactant production during this time period. The increase in PCconcentration during alveolarization is at least in part, and may beprimarily due to, a phospholipid composition defined aspalmitoylmyristoyl-PC (16:0/14:0PC) and not dipalmitoyl-PC (16:0/16:0PC)(FIG. 2 d).

Example 4 Phopholipid During Fetal and Early Postnatal Development

The present example demonstrates the utility of the present inventionfor use as a surfactant replacement preparation or as part of asurfactant replacement therapy in the treatment of IRDS or otherpulmonary function/developmental disorder, especially those related toprematurity and/or delayed/impaired alveolarization in infants.

The three predominant species of PC in surfactant (dipalmitoyl-PC(16:0/16:0PC), palmitoylpalmitoleoyl-PC (16:0/16:1PC) andpalmitoylmyristoyl-PC (16:0/14:0PC) ) were examined by absoluteconcentration and percentage distribution (FIG. 2) in animals bothbefore and after birth. Before birth, the proportion of dipalmitoyl-PC(16:0/16:0PC) increased from 23% at day 19 to 40% at birth (FIG. 2 d;Table 1). The proportion of larger acyl chain unsaturated (16:0/18:1PC,18:0/18:2PC, 16:0/20:4PC, 18:0/22:6PC and 18:1/18:2PC) PC declined(Table 1). TABLE 1 Content of PC in Fetal Rat BALF % Total PC PC SpeciesDay 19 Day 22 16:0/14:0 9.0 +/− 0.4 13.2 +/− 0.3*  16:0/16:1 14.1 +/−0.5  33.0 +/− 0.3*  16:0/16:0 22.9 +/− 0.4  38.8 +/− 0.5*  16:0/18:2 9.6+/− 0.7 2.8 +/− 0.1* 16.0/18:1 24.5 +/− 0.9  8.6 +/− 0.3* 18:1/18:2 4.0+/− 0.3 1.8 +/− 0.2* 18:0/18:2 3.8 +/− 0.1 0.5 +/− 0.0* 18.0/20:4 2.7+/− 0.2 0.6 +/− 0.1* 18:0/22:6 5.1 +/− 0.3 0.4 +/− 0.0**P ≦ 0.01. N = 4 separate BALF samples.

After birth the fractional concentration of dipalmitoyl-PC (16:0/16:0PC)remained consistently close to 40% (FIG. 2 d; Table 1), althoughabsolute values altered substantially (FIG. 2 a). These data indicatethat dipalmitoyl-PC(16:0/16:0PC) ratios with respect to total PC contentis regulated to a near constant value (FIG. 2 a). In some mammals,dipalmitoyl-PC (16:0/16:0PC) content in surfactant fluctuates between35%-60% [rabbits: 35.6% (6); humans: 54% (6, 33)]. This may define animportant concentration of 16:0/16:0PC for optimal surface-activefunction in vivo.

Surfactant from piglets is enriched in palmitoylpalmitoleoyl-PC(16:0/16:1PC) and palmitoylmyristoyl-PC (16:0/14:0PC) relative to adultpigs. The present data now demonstrates that these two PC species have adistinct profile during fetal and postnatal development (FIG. 2 b, 2 c,2 d). The fractional concentration of palmitoylpalmitoleoyl-PC(16:0/16:1PC) in BALF was greatest at birth (33%) and diminishedpostpartum (FIGS. 2 b,2 d; Table 1). There was no major change in itsconcentration between postnatal day 7 and 22 (FIG. 2 d).

The high concentration of palmitoylpalmitoleoyl-PC (16:0/16:1PC) atbirth (FIG. 2 b) may aid in the establishment of the first air/liquidinterface that is required at this time. This establishment requires arapid adsorption of surfactant to the interface. Besides surfactantproteins B and C (35), palmitoyloleoyl-PC (16:0/18:1PC) has also beenshown to improve dipalmitoyl-PC (16:0/16:0PC) adsorption at theair/liquid interface (11). Although palmitoyloleoyl-PC (16:0/18:1PC) mayimprove the adsorption rate of surfactant to the interface, it was foundthat its fractional concentration varied only little throughoutdevelopment. Moreover, its concentration was far below that ofpalmitoylpalmitoleoyl-PC (16:0/16:1PC) around birth (33% forpalmitoylpalmitoleoyl-PC (16:0/16:1PC) vs. 9% for palmitoyloleoyl-PC(16:0/18:1PC) ). Considering that palmitoylpalmitoleoyl-PC(16:0/16:1PC), like palmitoyloleoyl-PC (16:0/18:1PC), has a greatermolecule to water ratio at the air/liquid interphase than dipalmitoyl-PC(16:0/16:0PC) at a given pressure, it may have better adsorptioncharacteristics than dipalmitoyl-PC (16:0/16:0PC) (36-38). Therefore,palmitoylpalmitoleoyl-PC (16:0/16:1PC) is proposed to play an importantrole in forming the pulmonary surfactant film immediately after birth.

The palmitoylmyristoyl-PC (16:0/14:0PC) amount in BALF increased atbirth, consistently with the rise in concentrations of dipalmitoyl-PC(16:0/16:0PC) and palmitoylpalmi toleoyl-PC (16:0/16:1PC) (FIG. 2 c;Table 1). However, palrnitoylmyristoyl-PC (16:0/14:0PC) content in BALFpeaked between days 12 to 14 postpartum (FIG. 2 c). Fractionalpalmitoylmyristoyl-PC (16:0/14:0PC) concentrations were increased frompostnatal days 7 to 14 (FIG. 2 d), which corresponds to thealveolarization period in the rat (14). In fact, the general rise intotal PC content during this time (FIG. 1 aa) was primarily accountedfor by the increase in palmitoylmyristoyl-PC (16:0/14:0PC) (FIG. 2 d).

The function of palmitoylmyristoyl-PC (16:0/14:0PC) in pulmonarysurfactant is unclear. It has a similar molecule to water ratio at thewater/air interphase as dipalmitoyl phosphatidylcholine (16:0/16:0PC) ata given pressure (36, 37). The marginal chain asymmetry will likely notresult in a pronounced difference over dipalmitoyl-PC (16:0/16:0PC) withrespect to adsorption properties. Thus, it is unlikely thatpalmitoylmyristoyl-PC (16:0/14:0PC), in contrast topalmitoylpalmitoleoyl-PC (16:0/16:1PC) enhances the air/liquidadsorption rates of dipalmitoyl-PC (16:0/16:0PC). Considering theunclear role for palmitoylmyristoyl-PC (16:0/14:0PC) in surfactantfunction, additional work was done to determine whether the rise ofpalmitoylmyristoyl-PC (16:0/14:0PC) during alveolarization was specificto the rat. Therefore, BALF from mice was analyzed at late gestation(day 18) and around the peak (postnatal day 10) of alveolarization (15).

Mouse and rat BALF had similar fractional dipalmitoyl-PC (16:0/16:0PC)levels at fetal, postnatal (alveolar period) and mature time points(FIG. 3 a). Likewise, there was a similar trend between the two animalsfor high palmitoylpalmitoleoyl-PC (16:0/16:1PC) content in fetal samplesand high palmitoylmyristoyl-PC (16:0/14:0PC) content duringalveolarization (FIG. 3 b, 3 ca). This consistency between the tworodent species suggests that the relatively highpalmitoylpalmitoleoyl-PC (16:0/16:1PC) content of surfactant at birth aswell as the relatively high palmitoylmyristoyl-PC (16:0/14:0PC) contentof surfactant during alveolarization is a general phenomenon among allanimals, and therefore has application and utility in the treatment ofother animals, including humans.

Example 5 Surfactant Palmitoylmyristoyl Phosphatidylcholine(16:0/14:0PC) in Animal Models Having Compromised Alveolarization

The present example is presented to demonstrate the utility of thepresent invention as a marker useful in the detection of chemical anddisease-related damage to the lung. The relative concentration of the PCspecies, palmitoylmyristoyl-PC (16:0/14:0PC) is demonstrated herein tobe specifically correlated with the incidence of chemical-induced (suchas dexamethasone) damage to alveolar tissue and/or reducedalveolarization in an animal. Palmitoylmyristoyl-PC (16:0/14:0PC)concentration is also demonstrated to be a reliable indicator ofalveolar curvature and alveolar size in the animal lung.

Surfactant palmitoylmyristoyl-PC (16:0/14:0PC) increased during theperiod of alveolarization. To determine the nature of this relationship,BALF samples were examined from two rat models of diminishedalveolarization. The two models employed were postnatal exposure toeither dexamethasone or 60% oxygen. Postnatal administration ofdexamethasone to rats has been described to result in a reducedalveolarization (17, 39-41). In the present study, a high dose,short-term treatment with dexamethasone was used to induce reducedalveolarization in the animal. After this treatment at the age of 14days, a significant decrease in parenchymal complexity was observed inthe treated animals, with larger and fewer lung alveoli compared tocontrols (17).

Neonatal hyperoxia of mice and rats may also be used to inducediminished alveolarization (18, 42-44). In the present studies, exposureof neonatal rats to 60% oxygen was observed to result in a significantreduction of total PC in BALF at postnatal day 10 (FIG. 4 a). Bypostnatal day 14, total PC values were no longer significantly differentbetween 60% oxygen- and 60% O₂-exposed animals (FIG. 4 e).

Similar observations of reduced surfactant PC have been reported forneonatal rabbits exposed to 98% oxygen (45), although this effect couldbe the result of acute cellular injury. The content of individual PCmolecules, including dipalmitoyl-PC (16:0/16:0PC), palmitoylmyristoyl-PC(16:0/14:0PC) and palmitoylpalmitoleoyl-PC (16:0/16:1PC), were alsoreduced in BALF of rats exposed to 60% oxygen (FIG. 4 b-4 h).

The overall reduction in surfactant PC synthesis may be due to severalfactors. Hyperoxia is known to induce the release of a number ofcytokines (46). Some of these cytokines, in particular TNFα, have beenshown to reduce the activity of the rate limiting enzyme in PCsynthesis, CTP: phosphocholine cytidylyltransferase (47-50). Inaddition, oxidant stress has been shown to affect another crucial lipidsynthesizing enzyme, glycerol phosphate acyltransferase (51).Alternatively, hyperoxia increases lipid peroxidation in rat alveolartype II epithelial cells (52). Therefore, alveolar type II epithelialcells may be shunting their lipid production from surfactant synthesisto cellular membrane repair.

In contrast to hyperoxia, the present studies have demonstrated thatpost partum dexamethasone treatment results in a significant increase oftotal PC content in BALF at day 10 (FIG. 4 a). In particular,monounsaturated PC molecules, such as palmitoylpalmitoleoyl-PC(16:0/16:1PC) (FIG. 4 c) and palmitoyloleoyl-PC (16:0/18:1PC) (see FIG.7) were elevated in BALF of dexamethasone-treated neonatal rats. Similarobservations have been reported for liver PC of dexamethasone-treatedrats (53). Saturated PC species, including dipalmitoyl-PC (16:0/16:0PC),were also elevated in BALF of dexamethasone-treated neonatal rats (FIG.4 b) with the exception of palmitoylmyristoyl-PC (16:0/14:0PC), whichwas significantly reduced (FIG. 4 d). Palmitoylmyristoyl-PC(16:0/14:0PC) (FIG. 4 h) was the only PC species (FIG. 4 e-g)significantly reduced after 14 days of 60% oxygen exposure.

Thus, the two rat models of reduced alveolarization had contrastingeffects on surfactant PC production, i.e. up regulation withdexamethasone and down regulation with 60% O₂. With respect tosurfactant PC, a constant decrease was observed in palmitoylmyristoyl-PC(16:0/14:0PC) concentration. The results suggest that surfactantpalmitoylmyristoyl-PC (16:0/14:0PC) concentrations relate to thealveolarization process not only during normal lung development, butalso in two different models of diminished alveolar formation.

Example 6 Surfactant Palmitoylmyristoyl Phosphatidylcholine(16:0/14:0PC) Enriched Preparations and Pulmonary Disease

The present example is provided to demonstrate the utility of thepresent invention for use in detecting and/or diagnosing reducedpulmonary function and pulmonary disease in an animal, and particularlylung and/or alveolar related pathologies such as emphysema andbronchopulmonary dysplasia (BPD).

To evaluate the three predominant species of PC in human surfactant,tracheal aspirates were obtained from intubated neonates and BALF fromadult humans. Data was subdivided based on pathology. Tracheal aspirateswere obtained from infants with respiratory distress syndrome (RDS) whodeveloped BPD and from those infants who did not develop BPD.

BALF was obtained from adult human patients with emphysema or idiopathicpulmonary fibrosis (IPF), and from post lung transplant patients with nopronounced alveolar complication. These samples were compared with BALFobtained from lung cancer patients that had normal alveolararchitecture. Samples of emphysematic patients and infants who developedBPD displayed significantly reduced surfactant palmitoylmyristoyl-PC(16:0/14:0PC) levels compared to all other patient groups tested (FIG. 5b).

Dipalmitoyl-PC (16:0/16:0PC) did not significantly differ between thepatient groups (FIG. 5 a). Thus, the changes in palmitoylmyristoyl-PC(16:0/14:0PC) content in the emphysematic and BPD groups are likely notthe result of a general loss of surfactant. Emphysema is characterizedby abnormal, permanent enlargement of airspaces distal from the terminalbronchi, while one of the hallmarks of BPD seen in premature infants isalveolar simplification, i.e. larger but fewer alveoli. The reducedpalmitoylmyristoyl-PC (16:0/14:0PC) content in lavage fluid of BPD andemphysematic patients is in line with the rat models of reducedalveolarization. As such, the results suggest that surfactantpalmitoylmyristoyl-PC (16:0/14:0PC) content in humans also relate todistal airspace architecture of the lung.

From these studies, it is envisioned that a phospholipid compositioncomprising an enriched concentration of a surfactantpalmitoylmyristoyl-PC (16:0/14:0PC) will provide a pharmaceuticallyeffective lung surfactant therapy for patients having reducedalveolarization. Methods for treating an animal, such as a human, havingbeen diagnosed as having reduced alveolarization, such as ischaracteristic of patients having emphysema, BPD, IRDS, and pathologiesrelated thereto, are therefore provided by administering an effectiveamount of a pharmaceutically acceptable preparation of the surfactantpalmitoylmyristoyl-PC(16:0/14:0PC) as described herein.

In addition, a method of the present invention is provided foridentifying a patient having reduced alveolarization or other change inalveolar function/architecture related to disease, such as in emphysemaand BPD. The method, for example, would comprise obtaining a lung tissuesample, such as a tracheal aspirate or biopsy, and measuring the amountof palmitoylmyristoyl-PC (16:0/14:0PC) in the sample, wherein a reducedconcentration of palmitoylmyristoyl-PC (16:0/14:0PC) in the tissuesample compared to a control amount of palmitoylmyristoyl-PC(16:0/14:0PC) is diagnostic of reduced alveolar function or pulmonarydisease in the animal.

Example 7 Palmitoylmyristoyl Phosphatidylcholine (16:0/14:0PC) DuringAlveolarization and as an Indicator of Alveolar Curvature

The present example is provided to demonstrate the utility of theinvention as a pharmaceutically effective and specific lung surfactantpreparation useful for enhancing alveolarization and lung development inan animal. The present study was performed on tissue obtained fromrodents, and demonstrates the utility of the present preparations asuseful in the treatment of other animals, including humans.

The main feature of alveolarization is the subdivision of thepreexisting voluminous saccules by septation which leads to smallerunits (alveoli) and an increase in total surface area. These units thenhave the potential to increase in size during growth of the animal. Oneof the implications of saccular subdivision is an increase in alveolarcurvature. Two groups have reported alterations in alveolar diameter(14) and volume (54) during the period of alveolarization in the rat(14, 54, 55). When the calculated radii from the reported alveolardimensions were plotted against palmitoylmyristoyl-PC (16:0/14:0PC) inrat BALF during rat development, a striking relationship betweenpalmitoylmyristoyl-PC (16:0/14:0PC) content and alveolar curvature wasidentified by the present inventors (FIG. 6 a).

To even further characterize this relationship, light scatteringanalysis was performed on liposomes formed from total lipids extractedfrom BALF. Lipids in BALF of day 10 neonatal rats, which have a highsurfactant palmitoylmyristoyl-PC (16:0/14:0PC) content (FIG. 4 d),formed liposomes with an average particle size of 9.0±0.4 microns. Incontrast, lipids in BALF from dexamethasone and 60% oxygen-treated rats,which have a lower surfactant palmitoylmyristoyl-PC (16:0/14:0PC)content (FIG. 4 d), formed significantly larger liposomes (13.2±0.7microns and 12.6±0.6 microns, respectively). The BALF liposomes of 22day old rats (14±0.7 micron) were significantly larger than those of 10day-old rats consistent with the lower palmitoylmyristoyl-PC(16:0/14:0PC) content (FIG. 2 cd). When the radii of BALF liposomes wereplotted against BALF palmitoylmyristoyl-PC (16:0/14:0PC) content, astrong correlation (r₂=0.998) was found (FIG. 6 b). Thepalmitoylmyristoyl-PC (16:0/14:0PC) therefore serves to at least in partincrease the curving capacity of surfactant lipids. This would explain apotential basis for the increased palmitoylmyristoyl-PC (16:0/14:0PC)levels in surfactant of higher curved (smaller) air spaces that occursduring alveolarization. Other changes in variables such as surfactantproteins B and C may also be involved.

Intrinsic curvature in membranes can be influenced by small polar headlipids (56) or by asymmetric acyl chain length (57-59). Freeze fractureanalysis of PC has shown that dipalmitoyl-PC (16:0/16:0PC) liposomeshave a three-fold greater radius compared to palmitoylmyristoylphosphatidylcholine (16:0/14:0PC) liposomes (60). The acyl chains ofpalmitoylmyristoyl-PC (16:0/14:0PC) will not have the same surfacepacking as dipalmitoyl-PC (16:0/16:0PC) at 37° C. However, because ofits distinct acyl packing characteristics, it may obtain high surfacepressures when spread on a highly curved interface. Therefore,palmitoylmyristoyl-PC (16:0/14:0PC) improves surfactant function duringsecondary septation, which is associated with more curved surface areas.

Example 8 Alveolar Type II Epithelial Cells and PalmitoylmyristoylPhosphatidylcholine (16:0/14:0PC) Secretion During Lung Alveolarization

The present example is provided to demonstrate the correlation betweenalveolar Type II epithelial cell secretion of phosopholipid and earlypostnatal lung development.

Alveolar type II epithelial cells were found to play a role incontrolling the acyl composition of PC during alveolarization. Usinglaser capture microscopy and mass spectral lipid analysis,palmitoylmyristoyl-PC (16:0/14:0PC) content of rat alveolar type IIepithelial cells was found to increase postnatally from day 7; to peakat days 12-14 and to subsequently decline until day 21 when adult levelswere reached (FIG. 6 c). This profile for the increase and decrease inpalmitoylmyristoyl-PC (16:0/14:0PC) content was also observed inbronchoalveolar lavage samples examined from animals at the samecorresponding developmental time periods.

The similarity in the palmitoylmyristoyl-PC (16:0/14:0PC) contentprofiles from both the cellular (alveolar Type II epithelial cells) andbronchoalveolar lavage (BALF) samples during the alveolarization perioddemonstrates that lipid changes in the BALF are due to alveolar type IIepithelial cells producing a different surfactant. How the alveolar typeII epithelial cells sense architectural changes and produceacyl-specific PC during alveolarization remains to be elucidated.

Example 9 Palmitoylmyristoyl Phosphatidylcholine (16:0/14:0PC) EnrichedPreparations for Treatment of Lung (Alveolar) Damage associated withChemical Exposure

The present example demonstrates the utility of the invention in thetreatment of pulmonary disease or damage resulting from pulmonaryexposure to potentially toxic or otherwise damaging agents. It isenvisioned that chronic exposure to commonly used inhalable preparationsof steroids, such as those used in the treatment of asthma, results inalveolar/pulmonary damage that may be detected and treated using thesurfactants, surfactant supplements, and pulmonary disease markers ofthe present invention.

The method for the prevention or treatment of pulmonary disease ordestruction consequent exposure to chemical and steroidal elements isprovided comprising introducing a phosopholipid preparation enriched fora phospholipid palmitoylmyristoyl-PC (16:0/14:0PC), alone or incombination with other active ingredients (such as Protein B, C, D, orother protein), in an amount effective to reduce the symptoms of orprevent pulmonary disease, wherein the pulmonary disease is reactiveoxygen-induced or mediated pulmonary damage, chemically induced lunginjury, injury due to oxygen radicals, injury due to ozone, injury dueto chemotherapeutic agents, inflammatory and infectious diseases,reperfusion injury, drowning, lung transplantation, and organ (lung)rejection.

Example 10 Preparations having Enriched 16:0/14:0PC Concentrations withPulmonary Surfactant Proteins

The surfactant formulations of the present invention comprise in someembodiments an enriched concentration of the PC,palmitoylmyristoyl-PC(16:0/14:0PC). These preparations may be formulatedtogether with one or more important pulmonary surfactant proteins. Suchpulmonary surfactant proteins include, by way of example, PulmonarySurfactant protein A (SP-A), Pulmonary Surfactant Protein B (SP-B),Pulmonary Surfactant Protein C (SP-C) and Pulmonary Surfactant Protein D(SP-D).

These preparations may take the form of a lung “wash” (for use as alavage), or may be formulated in an aerosol.

Combination with Pulmonary Surfactant Protein B (SP-B)—

It is envisioned that the palmitoylmyristoyl-PC (16:0/14:0PC)-enrichedpreparations of the present invention may be formulated to include aneffective amount of the naturally occurring human surfactant Protein B,or a fragment thereof (e.g., N-terminal end fragment).

Naturally occurring SP-B has a length of 78 amino acid residues, anN-terminal residue of phenylalanine and a simple molecular weight ofabout 8,700. SP-B isolated from human lung migrates on polyacrylamidegels as an entity having a relative molecular weight (Mr) of 7-8,000after sulfhydryl reduction. Without sulfhydryl reduction, the naturallyoccurring protein is also found as large oligomers. SP-B is hydrophobic,which is consistent with its in vivo strong association withphospholipids and solubility in organic solvents such as chloroform andmethanol.

A porcine-derived Surfactant Protein B (about 0.2 mg/ml (0.2-0.4 mg/ml),extracted from porcine lungs, has been described in combination withDPPC (31 mg/ml), to form an intratracheal suspension, CUROSURF®. Theformulations of the present invention in some embodiments are notenvisioned to include porcine-derived SP-B. The present formulationswould include an enriched concentration of 16:0/14:0PC, and similarconcentrations of synthetic SP-B or porcine-derived SP-B may also beincluded.

Synthetic forms of Pulmonary Surfactant Protein B have been described inU.S. Pat. No. 6,660,833, U.S. Pat. No. 6,838,428 and U.S. Pat. No.5,547,937. One example of a Protein-B based pulmonary preparation isLucinactant.

A particular synthetic form of human Protein B is known as KL4. KL4(also known as sinapultide) mimics the attributes of human SP-B. NativeSP-B in natural pulmonary surfactant functions in surface tensionlowering and promoting oxygen exchange. Chemically, KL4 consists of 21amino acid residues where “K” is the amino acid lysine and “L” is theamino acid leucine. KL4-surfactant is an aqueous suspension consistingof KL4, the lipids DPPC and palmitoyloleoyl phosphatidylglycerol (POPG),plus the fatty acid, palmitic acid (PA).

Combination with Pulmonary Surfactant Protein C (SP-C)

It is envisioned that the palmitoylmyristoyl-PC (16:0/14:0PC)-enrichedpreparations of the present invention may be formulated to include anamount of the naturally occurring human surfactant Protein C, or afragment thereof.

A synthetic (recombinant) Surfactant Protein C is described in U.S. Pat.No. 5,876,970. Native SP-C has an amino terminal glycine residue, amolecular weight of about 3,700, a polyvaline sequence, and is extremelyhydrophobic. It is also substantially resistant to enzyme degradation byproteases (trypsin, chymotrypsin and staphylococcus nucleotide V-8),endoglycosidase F, and collagenase.

A calf-derived phospholipid preparation, INFASURF® (calfactant), hasalso been described. This preparation contains a natural surfactant fromcalf lungs including phospholipids (35 mg total phospholipids, including26 mg phosphatidylcholine, of which 16 mg is disaturatedphosphatidylcholine), neutral lipids, and hydrophobicsurfactant-associated protein-B and C (SP-B and SP-C, 0.65 mg protein,including 0.26 mg of SP-B).

The formulations of the present invention in some embodiments mayinclude calf-derived phospholipids, or may be formulated to primarilyinclude synthetic, non-animal derived phospholipids. In someembodiments, the present inventive formulations would include anenriched concentration of palmitoylmyristoyl-PC (16:0/14:0PC), with orwithout synthetic SP-B and SP-C.

A bovine lung tissue extract prepared from minced calf lungs, has alsobeen described that contains bovine phospholipids. One such preparationis SURVANTA®. The formulations of the present invention in someembodiments are envisioned to include bovine-lung derived phospholipids,or to instead include non-bovine lung, synthetic phospholipids. In someembodiments, the present inventive formulations would include anenriched (at least 50% by weight or more total phospholipid)concentration of palmitoylmyristoyl-PC (16:0/14:0PC).

Combination with Pulmonary Surfactant Protein D (SP-D)

It is envisioned that the palmitoylmyristoyl-PC (16:0/14:0PC)-enrichedpreparations of the present invention may be formulated to include aneffective amount of the naturally occurring human surfactant Protein D,or a fragment thereof.

A synthetic (recombinant) Surfactant Protein D is described in U.S. Pat.No. 6,838,428. Native SP-D is a 43-kDa member of the collectin family ofcollagenous lectin domain—containing proteins that are expressed inepithelial cells of the lung. Synthetic forms of SP-D are described inLu et al., Purification, “Characterization and cDNA cloning of HumanLung Surfactant Protein D”, (1992), Biochem. J. 284: 795-802, which isspecifically incorporated herein by reference. Protein D is alsodescribed in U.S. Pat. No. 6,838,428, the teachings of which are alsospecifically incorporated herein by reference.

SP-D is associated with anti-pulmonary viral activity, and therefore isenvisioned as particularly suitable for use in the compositions of thepresent phospholipid PC preparations to be administered to animalsafflicted with some form of pulmonary viral disease, such as emphysema.

Combination with Colfosceril Palmitate (DPPC)

It is envisioned that the palmitoylmyristoyl-PC (16:0/14:0PC)-enrichedpreparations of the present invention may be formulated to include anamount of the phospholipid, colfosceril palmitate (commonly known asDPPC).

Colfoscheril palmitate has been included as a major component ofpreparations suitable as intratracheal suspensions that areprotein-free. One such preparation formulated for use in infants isEXOSURF NEONATAL®. In suspension, EXOSURF NEONATAL® includes 13.5 mg/mlcolfosceril palmitate, 1.5 mg/ml alcohol, and 1 mg/ml tyloxapol in 0.1 NNaCl. This preparation suspension is typically given directly to thelung through a tube (endotracheal administration).

It is envisioned that the palmitoylmyristoyl-PC (16:0/14:0PC)-enriched(about 50% by weight or more) preparations of the present invention maybe formulated to include an effective amount of other species ofphospholipids, such as DPPC, for example.

Example 11 Preparations having Enriched 16:0/14:0PC Concentrations

The present example defines the preparation as formulated from syntheticphospholipid sources.

The synthetic surfactant preparation for administration to prematureinfants includes about 30 mg/ml phosphatidylcholines comprising about30% (12 mg/ml) palmitoylpalmitoleoyl-PC (16:0/16:1PC) and about 50% (15mg/ml) dipalmitoyl-PC (16:0/16:0PC), about 20% (6 mg/ml)palmitoylmyristoyl-PC (16:0/14:0PC), about 0.2 to about 0.4 mg/mlsynthetic SP-B, and about 0.5 mg synthetic SP-D, or is a natural adult(porcine or calf) surfactant enriched with palmitoylpalmitoleoyl-PC(16:0/16:1PC) and palmitoylmyristoyl-PC (16:0/14:0PC) to an endconcentration of at least about 30% and about 20%, respectively (seeFIG. 8).

The synthetic surfactant preparation for administration to emphysematicpatients, in some embodiments, includes about 30 mg/mlphosphatidylcholines comprising about 20% (6 mg/ml)palmitoylpalmitoleoyl-PC (16:0/16:1PC), about 50% (15 mg/ml)dipalmitoyl-PC (16:0/16:0PC), about 30% (9 mg/ml) palmitoylmyristoyl-PC(16:0/14:0PC), about 0.2 to about 0.4 mg/ml synthetic SP-B and about 0.5mg/ml synthetic SP-D. In other embodiments, the synthetic surfactantpreparation comprises a natural adult (porcine or calf) surfactantenriched with palmitoylpalmitoleoyl-PC (16:0/16:1PC) (at least about20%) and palmitoylmyristoyl-PC (16:0/14:0PC) (at least about 30%), andabout 0.5 mg/ml synthetic SP-D.

Example 12 b 16:0/14:0PC as a Marker to Predict Risk in Infants forContinued Respiratory Distress

The present example is provided to demonstrate the utility of theinvention as a method for predicting risk in a premature infant withrespiratory distress for future development of bronchopulmonarydysplasia. By employing palmitoylmyristoyl-PC (16:0/14:0PC) as a markerin samples from premature infants with respiratory distress, one caneffectively predict with a high degree of accuracy the percentage ofthese infants that will continue to suffer from pulmonary compromisedconditions (see FIG. 9).

As shown in FIG. 9, a significant number of human infants diagnosed withRDS that went on to develop BPD (CLD) had an 16:0/14:0PC content (%) of7% or less. Human infant samples taken from infants that had RDS and hadalready been diagnosed with BPD also had a 16:0/14:0PC content (%) of 7%or less. In contrast, samples obtained from human infants diagnosed withRDS that did not develop BPD (CLD) had a 16:0/14:0PC content (%) of morethan about 7%. Hence, a reduced relative concentration percent (%) of16:0/14:0PC (such as for example, about 10%, 20%, 25% or as much as 40%less 16:0/14:0PC compared to a control infant lung sample) in an infantlung sample is a prognostic indicator for identifying an infant at riskfor developing BPD (CLD).

All documents, patents, journal articles and other materials cited inthe present application are hereby incorporated by reference.

Although the present invention has been fully described in conjunctionwith several embodiments thereof with reference to the accompanyingdrawings, it is to be understood that various changes and modificationsmay be apparent to those skilled in the art. Such changes andmodifications are to be understood as included within the scope of thepresent invention as defined by the appended claims, unless they departthere from.

BIBLIOGRAPHY

The following references are specifically incorporated herein byreference.

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1. A phospholipid preparation suitable for use as a lung surfactant orlung surfactant supplement comprising an enriched concentration ofpalmitoylmyristoyl-PC (16:0/14:0PC) in a pharmacologically acceptablecarrier solution.
 2. A marker of reduced alveolarization or pulmonarydisease state in a subject animal comprising a subject animal samplepalmitoylmyristoyl-PC (16:0/14:0PC) concentration.
 3. The marker ofclaim 2 wherein a reduced alveolarization or pulmonary disease state inthe subject animal is indicated by a reduced subject animal samplepalmitoylmyristoyl-PC (16:0/14:0PC) concentration.
 4. The marker ofclaim 3 wherein the subject animal lung sample palmitoylmyristoyl-PC(16:0/14:0PC) concentration is reduced about 20% relative to a controlanimal lung sample palmitoylmyristoyl-PC (16:0/14:0PC) concentration asdetermined by mass spectral analysis.
 5. The marker of claim 4 whereinthe subject animal lung sample palmitoylmyristoyl-PC (16:0/14:0PC)concentration is reduced about 25% relative to the control animal lungsample palmitoylmyristoyl-PC (16:0/14:0PC) concentration as determinedby mass spectral analysis.
 6. A method for treating an animal having acompromised pulmonary condition comprising administering apharmacologically effective amount of a surfactant preparation having anenriched concentration of palmitoylmyristoyl-PC (16:0/14:0PC) relativeto other phospholipids in a pharmacologically acceptable carrier.
 7. Themethod of claim 6 wherein the surfactant preparation ofpalmitoylmyristoyl-PC (16:0/14:0PC) comprises an enriched concentrationof more than 50% of the phospholipids of the preparation.
 8. The methodof claim 6 wherein the surfactant preparation is essentially free ofphospholipids other than palmitoylmyristoyl-PC (16:0/14:0PC).
 9. Amethod for identifying a subject having reduced alveolarization or otherchange in alveolar function/architecture related to disease or exposureto a toxic substance comprising; obtaining a tissue lung sample from asubject animal to provide a subject lung sample; measuring an amount ofpalmitoylmyristoyl-PC (16:0/14:0PC) in the subject lung sample; andcomparing the amount of palmitoylmyristoyl-PC (16:0/14:0PC) in thesubject lung sample to an amount of palmitoylmyristoyl-PC (16:0/14:0PC)in a control lung sample obtained from a control animal, wherein areduced concentration of palmitoylmyristoyl-PC (16:0/14:0PC) in thesubject lung sample compared to the amount of palmitoylmyristoyl-PC(16:0/14:0PC) in the control lung sample is diagnostic of reducedalveolar function or pulmonary disease in the subject animal.
 10. Themethod of claim 9 wherein the reduced alveolar function related todisease is emphysema, respiratory distress syndrome, idiopathicpulmonary fibrosis, broncho pulmonary dysplasia or diseases with primarydefects of alveolarizaion.
 11. The method of claim 9 wherein the amountof palmitoylmyristoyl-PC (16:0/14:0PC) in the subject lung sample isreduced at least 20% compared to the amount of palmitoylmyristoyl-PC(16:0/14:0PC) in the control lung sample.
 12. The method of claim 9wherein the subject lung sample has an elevated amount of totalphospholipids compared to the total amount of phospholipids of a controllung sample.
 13. The method of claim 12 wherein the total phospholipidscomprise palmitoylpalmitoleoyl-PC (16:0/16:1PC) and palmitoyloleoyl-PC(16:0/18.1PC).
 14. The method of claim 9 wherein the subject lung samplehas an elevated amount of dipalmitoyl-PC(16:0/16:0PC) relative to theamount of dipalmitoyl-PC (16:0/16:0PC) in the control lung sample. 15.The method of claim 14 wherein the subject lung sample has an elevatedamount of dipalmitoyl-PC (16:0/16:0PC) of about 20% to about 40% greaterthan the amount of dipalmitoyl-PC(16:0/16:0PC) in the control lungsample.
 16. The method of claim 14 wherein the subject lung sample hasan elevated amount of dipalmitoyl-PC (16:0/16:0PC) of about 28% to about33% greater than the amount of dipalmitoyl phosphatidylcholine(16:0/16:0PC) in the control lung sample.
 17. The method of claim 9wherein the subject lung sample has an elevated amount ofpalmitoylpalmitoleoyl-PC (16:0/16:1PC) compared to the amount ofpalmitoylpalmitoleoyl-PC (16:0/16:1PC) in the control lung sample.
 18. Asurfactant pretreatment suitable for inhibiting lung damage comprising apreparation enriched for palmitoylmyristoyl-PC (16:0/14:0PC).
 19. Thesurfactant pretreatment of claim 18 suitable for reducing lung damageattendant lung-ischemia-reperfusion injury.
 20. A method for reducinglung damage attendant lung ischemia-reperfusion in a patient having alung transplant comprising pretreating the patient with the surfactantpretreatment of claim 18.